PURIFICATION AND CHARACTERIZATION OF AMINOPEPTIDASE-P FROM LACTOCOCCUS-LACTIS SUBSP CREMORIS

Aminopeptidase P was purified 65.3-fold from the cytoplasm of Lactococ cus lactis subsp. cremoris AM2 with a 5.8% yield. The purified enzyme was found to consist of one polypeptide chain with a relative molecula r mass of 41 600. Metal chelating agents were found to be inhibitory a nd Mn2+ and Co2+ stimulated activity 7-fold and 6-fold respectively. T he purified enzyme removed the N-terminal amino acid from peptides onl y where proline (and in one case alanine) was present in the penultima te position. No hydrolysis was observed either with dipeptides even wh en proline was present in the C-terminal position or when either N-ter minal proline or pyroglutamate was present preceding a proline residue in the penultimate position of longer peptides. On the basis of this substrate specificity either aminopeptidase P or post-proline dipeptid yl aminopeptidase are necessary along with a broad specificity aminope ptidase to effect complete hydrolysis of casein-derived peptides conta ining a single internally placed proline residue. However, both aminop eptidase P and post-proline dipeptidyl aminopeptidase would be require d together with a broad specificity aminopeptidase in order to complet ely hydrolyse casein-derived peptides that contain two internally plac ed consecutive proline residues. As bitter casein-derived peptides are likely to contain either single prolines or pairs of prolines, aminop eptidase P appears to be an important enzyme for debittering.